Cancer cells have developed at least three mechanisms to evade a host's immune surveillance: 1) evasion of immune surveillance by T-lymphocytes, by high expression of membrane protein programmed death-ligand 1 and programmed death-ligand 2 (PD-L1 and PD-L2), both of which bind to programmed cell death protein (PD-1) on the surface of T-cell, inducing T-cell apoptosis. 2) evasion of immune surveillance by natural killer (NK) cells. The NKG2D protein on the surface of NK cells, upon binding to the MICA/MICH proteins on the surface of the cancer cells, can activate NK cells which can then kill the cancer cells. However, cancer cells have developed a mechanism that promotes the detachment of MICA/MICB from the cancer cells. The detached MICA/MICH binds to the NKG2D, blocking its activation of the NK cells. 3) Evasion of the immune surveillance of macrophages (Mφ). Almost all cancer cells express on their surface a high level of Cluster of Differentiation 47 (CD47)[1], which is also known as integrin associated protein (IAP). CD47 binds to the signal regulatory protein α(SIRPα) on the surface of Mφ, thereby inducing the production of an inhibitory signal, which inhibits the phagocytosis function of Mφ[2]. Development of effective anti-cancer drugs needs to target these mechanisms[1].
In addition, growth of cancer cells depends on sufficient supply of nutrition. Cancer cells themselves can secrete factors that promote blood vessel growth, such as vascular epithelial growth factors (VEGF). Inhibition of the activity of VEGF will stop blood supply to the tumor, thereby inhibiting the growth of cancer. For example, a drug sold under the tradename AVASTIN™, an FDA approved antibody drug, functions to treat cancers (colon cancer, lung cancer) by inhibiting the biological activities of VEGF. Another protein drug, sold under the trade name ZALTRAP™, which was approved for marketing in August of 2012, for treating colon cancer, also targets VEGF. However, these drugs only inhibit cancer cell growth to certain extent, and cannot eliminate the cancer cells.
Signal Regulatory Protein is a family of trans-membrane proteins, with three members: SIRPα (CD172a), SIRPβ (CD172b), SIRPγ (CD172g). All three members comprise a similar extracellular region, but different intracellular domains. The extracellular domain comprises three Ig-like regions, where the first Ig-like region is an Ig-V region, while the second and third regions are IG-C regions.
The intracellular domain of SIRPα (CD172a) contains two inhibitory signaling regions that can inhibit signal transduction and corresponding cell functions. The intra-membrane domains of SIRPβ (CD172b) and SIRPγ (CD172g) are very short, and do not contain a signal transduction region, but SIRPβ (CD172b) may function through an adaptor protein, e.g. DAP12 for signal transduction (see FIG. 1). SIRPs are primarily expressed on macrophages (Mφ), dendritic cells (DC) and neurons.
CD47 is also a transmembrane glycoprotein belonging to the immunoglobulin superfamily, and is expressed on the surface of all cell types including red blood cells. Ligands for CD47 include integrins, thrombospondin-1 and SIRPs. CD47 has many biological functions, including in cell migration, activation of T-cells and DCs, and neural development. In addition, CD47, by interacting with SIRPα, can inhibit the phagocytosis of macrophages. By emitting a “do not attack” signal, CD47 protects normal cells, such as blood cells, from being attacked by macrophages.
As mentioned above, many tumor or cancer cells over-express CD47, which, by binding to the SIRPα on the cell surface of macrophages, prevent phagocytosis of the cancer cells by macrophages. Cancer cells that over-express CD47 include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-hodgkins lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, and pancreatic cancer. Injection of tumor-bearing mice with CD-47 specific antibody can significantly inhibit tumor growth in vivo[3-4]. Cancer cells in mice carrying human leukemia cells were eliminated completely when the same antibody was injected into the mice[5].
VEGFs are a family of secreted glycoproteins, with a molecular weight of about 40 kDa. This family has 5 members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF. There are three VEGF receptors (VEGFRs), including VEGF VEGFR, and VEGFR3, each of which can bind selectively with different ligands. For example, VEGFR1 binds with VEGF-A, VEGF-B, and PIGF, VEGFR2 binds with VEGF-A, VEGF-C, and VEGF-D, but not PIGF; VEGFR3 binds only with VEGF-C and VEGF-D. The receptors have different functions. VEGFR1 binds to VEGF-A with very high affinity, 10 times higher than VEGFR2, so it functions to negatively regulate VEGFR2. VEGFR2 induces vascular endothelial cell growth, promoting growth of blood vessels. VEGFR3, on the other hand, is related to lymphatic duct development and growth. Of all VEGFs and their receptors, it can be said that VEGF-A and VEGFR2 are the most important, as in certain disease states, (e.g. cancer and age-related macular degeneration), over secretion of VEGF-A, which then binds to VEGFR2, will cause abnormal vascular growth, resulting in disease development or aggravation. Therefore, VEGF-A and VEGFR2 are both important drug targets.
An anti-VEGF monoclonal antibody, sold under the tradename BENAVIZUMAB™, was approved for marketing in 2004, and is indicated for metastatic colon cancer, lung cancer and renal cancer. A recombinant protein drug sold under the tradename ZALTRAP™, also known as VEGF-Trap, which also targets VEGF, was approved for marketing to treat metastatic colon cancer in 2012. Another antibody drug under the tradenaine RAMUCIRUMAB™, targeting VEGFR2, is undergoing a phase III clinical trial. No anti-CD47 drug is on the market, and all are in pre-clinical stage.
There is no report so far of any single molecule drug that targets both CD47 and VEGF, and the present invention satisfies this need.